Single Chain Fv constructs of anti-ganglioside GD2 antibodies

ABSTRACT

Recombinant antibody constructs comprise the variable regions of the heavy and light chains of anti-G D2  antibodies. These antibody constructs may be coupled to a label such as a radiolabel or to a protein such as streptavidin or pro-drug converting enzymes for use in imaging or therapeutic applications. The antibody constructs may also be transduced into T cells to produce populations of T cells which target G D2 -producing tumor cells.

BACKGROUND OF THE INVENTION

[0001] This application relates to single chain antibody constructswhich specifically bind to the disialoganglioside G_(D2), and to the useof such constructs for targeted delivery of imaging agents ortherapeutic agents to human neuroectodermal derived cancers.

[0002] Gangliosides are acidic glycosphingolipids found on the outersurface of most cell membranes.¹ Many tumors have abnormal glycolipidcomposition and structure. Disialoganglioside G_(D2) has been found in awide spectrum of human tumors, including neuroblastoma, osteosarcomasand other soft tissue sarcomas, medulloblastomas, high gradeastrocytomas, melanomas, and small cell lung cancer.²⁻⁴ Amongglioblastoma multiforme and anaplastic astrocytoma, anti-G_(D2)demonstrated the most restrictive pattern when compared with anti-GD3and anti-GM2 antibodies.^(5,6)

[0003] Gangliosides are ideal targets for monoclonal antibodies (MAb)because of the high antigen density, lack of modulation, relativehomogeneity in many tumors and the possibility of up-regulation bycytokines.⁷ The only normal tissues with high ganglioside expression areneurons, and biodistribution studies have shown that MAb do not localizeto the nontumorous brain or spinal cord because of the blood brainbarrier. In contrast, in patients with primary or metastatic braintumors, specific antibodies can localize preferentially to tumortissues, but not to normal brain.⁸

[0004] Murine monoclonal antibodies have been prepared to gangliosideG_(D2) Using somatic cell hybridization, murine MAbs were producedagainst the ganglioside GD₂.⁹ They were shown to react withdisialoganglioside G_(D2), but not with GD3, GT1b, GD1b, GD1a, GM1, GM3and GM4. When base-treatment step was omitted from the standardneuroblastoma ganglioside extraction procedure,immuno-thin-layer-chromatography (ITLC) using 3F8, 3G6 and otheranti-G_(D2) MAbs revealed a new ganglioside band with Rf of 0.342,besides G_(D2) (Rf 0.183).⁴ Immunochemical analysis showed that this newneuroblastoma ganglioside contained alkali-sensitive O-acetylated sialicacid residues recognized by MAb D1.1.

[0005] Of 15 anti-G_(D2) MAbs studied, 13 reacted strongly with thenovel ganglioside. 3F8 was chosen for our initial clinical studiesbecause of its being an IgG3 and its strong binding in vitro to G_(D2).Based on the cDNA sequence and the anti-idiotype cross-reactivity, theantigen specificity and affinity of 3f8 and 3G6 were similar if notidentical. We have chosen 3G6 for scFv development for ease ofcomparison with the other 14 MAbs which are IgM antibodies. TABLE 1SPECIFICITY OF ANTI-G_(D2) MAb. MAb G_(D2) GD3 “O-G_(D2)” GD1b 1A8 4+ −+/− 1+ 1F9 4+ +/− 3+ 1+ 1H12 4+ +/− 3+ 1+ 2F7 3+ − 1+ — 3A7 3+ − − —3A10 4+ 1+ 2+ 1+ 3B4 4+ − 3+ 1+ 3F8 4+ − 4+ — 3G6 4+ − 2+ — 4C11 4+ − 3++/− 5E11 4+ − 4+ — 5F4 4+ 1+ 2+ 1+ 5F11 2+ +/− 3+ 1+ 6E8 4+ 1+ 3+ 1+ 6H44+ +/− 3+ 1+

[0006] In order to determine the general applicability of theganglioside G_(D2) as a target for immunotherapy, its expression inhuman cancers has been studied by imnmunostaining tumor specimens usingthese monoclonal antibodies. These anti-G_(D2) antibodies reacted withall the neuroblastoma surgical specimens tested to date in ourlaboratory. A recent update¹⁰ analyzed a series of 39 neuroblastomas.Staining of both primitive neuroblastic and differentiatingganglioneuromatous elements were seen, although tumor cell heterogeneitywas noted in some. 23/39 tumors showed a more intense reactivity withMAb 3A7 than with 3F8, and this was particularly evident in theprimitive neuroblastoma group. In a separate study, the expression ofG_(D2) was analyzed in 67 solid tumors and normal tissues from childrenby using the antibody 3A7.¹¹ G_(D2)expression was found in 28 of 28neuroblastomas, and was most abundant in stroma-poor tumors.Differentiating stroma-rich neuroblastomas, neuroblastic clusters,neurofibrils, and most ganglion-like cells were found to be G_(D2)positive, whereas Schwann's-cell stroma did not express G_(D2). Inganglioneuromas, only a few ganglion-like cells showed G_(D2), whereasall other structures were negative. Scattered foci of G_(D2) were alsofound in some non-neuronal tumors, such as rhabdomyosarcomas andosteosarcomas, but not in lymphomas, Askin tumors, or most Wilm'stumors. 3A7 was also found to react with retinoblastomas.¹²

[0007] Previous studies have shown that anti-G_(D2) antibodies reactedwith the majority of osteosarcomas.¹³ Sixty freshly frozen humansoft-tissue sarcomas were studied by avidin-biotin immunostaining usingpurified monoclonal antibodies 3F8 (anti-G_(D2)) and R24(anti-G_(D3)).¹⁴ Ninety-three percent of the tumors tested by theimmunohistochemical staining expressed G_(D2) and 88% expressed GD3. Theintensity of expression varied among different histologic types.Liposarcoma, fibrosarcoma, malignant fibrous histiocytoma,leiomyosarcoma and spindle cell sarcoma reacted strongly with bothantibodies. Embryonal rhabdomyo-sarcoma and synovial sarcomademonstrated substantially weaker staining by either MAb. Gangliosideextraction and immuno-thin layer chromatography (ITLC) confirmed theidentities of these gangliosides as G_(D2) and GD3 respectively

[0008] Among brain tumors, 3F8 and 3A7 have also shown excellentreactivities. Two separate studies were carried out: the first study incollaboration with Dr. Paul Zeltzer of Texas and the second with Dr. IraBergman (now Associate Professor of Neurology at the University ofPittsburgh) in our laboratory. In the first study, 12/15 medulloblastomaand 16/18 astrocytoma were positive, the majority staininghomogeneously. In the second study, similar results were obtained.Medulloblastoma and a number of brain tumors reacted strongly with 3F8and 3A7. The pattern of reactivity was generally homogeneous. For smallcell lung cancer, all have reacted homogeneously in vitro usingimmunoperoxidase techniques.

[0009] Despite in vitro evidence for exquisite specificity of theseantibodies for the ganglioside G_(D2) on neuroblastoma cells, a criticaltest of in vivo delivery is the actual amount of MAb uptake in thetumors. Biodistribution of 131I-anti-G_(D2) antibody was tested inpreclinical experiments using athymic mice xenogratted with humanneuroblastoma.

[0010] Between 8 to 50% injected dose of 131I-MAb/gm of tumor was found,with variability depending primarily on the size of the tumor.¹⁵ Therewas no localization to G_(D2)-negative tumors like Ewing's sarcoma.Pooled mouse IgG and an irrelevant MAb also did not localize toneuroblastoma xenografts. Both small tumors (50 mg) and large tumors(over 2 g) showed radiolocalization with this technique. Optimal tumorto normal tissue ratios were rapidly reached by 24 to 48 hours. Therewas no increased uptake in the reticuloendothelial system, and the MAbdid not cross the intact blood-brain barrier. The efficacy of tumortargeting was then tested by imaging neuroblastoma patients with131I-MAb. Radiolocalization was demonstrated in primary tumors of themediastinum and abdomen, as well as metastatic disease in the lymphnodes, bone marrow and bone.^(16,17) The specificity was validated bytumor and marrow biopsies, as well as by CT/MRI and bone scans. Acomparison with 131I-meta-iodobenzylguanidine (MIBG) suggested that131I-MAb was twice as sensitive in detecting metastatic sites ofdisease. The tumor uptake in patients was 0.08% of the injected dose pergm (compared to 0.002% for MIBG). This high tumor uptake in vivo was aresult of (1) the high density (5×106/cell) and homogeneity of thetarget antigen G_(D2), and (2) the lack of uptake in thereticuloendothelial system. A number of human cancers has been imagedusing G_(D2) specific antibodies. These include small cell lungcancer,¹⁸ brain tumors,⁸ and both osteosarcomas¹³ and soft tissuesarcomas.¹⁹

[0011] A phase I study to test the biological toxicity of “cold”anti-G_(D2) was carried out in 1987 in 17 patients with metastaticneuroblastoma or melanoma. A subsequent phase II study was carried outin 16 patients with stage IV neuroblastoma. Acute self-limitedtoxicities of MAb treatment were severe pain requiring analgesics,fever, urticaria, hypertension, hypotension, anaphylactoid reactions ofthe respiratory tract, as well as significant decreases in blood countsand serum complement levels. There were no treatment related deaths.Among the 5 neuroblastoma patients who are still alive and well (19 mos,3y, 5y, 5y, 6y respectively after MAb treatment), there are no acute ordelayed neurological complications attributable to MAb therapy. Amongthe survivors, one patient had chemo-radiotherapy-resistant stage IVSneuroblastoma, and the other 4 had poor risk stage IV neuroblastomadiagnosed at more than one year of age (2 relapsed neuroblastoma and 2with refractory neuroblastoma prior to antibody treatment).

[0012] More recently, a phase I study to determine the radiologicaltoxicity was carried out. Twenty-three patients (11 M and 21 F, rangingfrom 0.3 to 24.2 years of age at diagnosis) with refractoryneuroblastoma (22 stage IV, 1 stage IIIU), were treated with 131I-3F8 at7 dose levels, namely 6, 8, 12, 16, 20, 24, and 28 mCi/kg. Radiationdose to the blood was calculated based on blood clearance; total bodydose was based on total body clearance, and the tumor/organ dose onregions of interest calculations from serial gamma imagings. 21/23patients were rescued with autologous bone marrow; one patient receivedGM-CSF alone, one died of progressive disease before marrow reinfusion.Marrow was infused when blood radioactivity decreased to <0.01 uCi/ml inthe first 18 patients and to <1 uCi/ml in the last 4 patients. Acutetoxicities of 131I-MAb treatment included pain (19/23) during theinfusion, fever (19/23), hyperbilirubinemia (6/23), and diarrhea. Allpatients developed grade 4 myelosuppression with sepsis in 7/23 patients(5 fungal, 2 bacterial), disseminated zoster in 1, and pneumocystisin 1. Despite orally administered saturated solution of potassiumiodide, 3 patients developed hypothyroidism. Subsequent 14 patients weretreated with synthroid or Cytomel for thyroid protection. No othersignificant extramedullary toxicities have been encountered in patientsfollowed past 20 months (50+, 40+, 30+, 26+, 23+, mos) from the time of131I-MAb treatment. Fourteen patients have died 11 of disease and 3 frominfections during the cytopenic period, and in 4 patients follow-up isstill short. Responses were seen in both soft tissue masses and bonemarrow. Average tumor dose was 150 rad/mCi/kg. We concluded that when131I-MAb was administered intravenously (6-28 mCi/kg), significanttoxicities were encountered, including myelosuppression and theirinfectious complications, pain, fever, as well as hypothyroidism.Autologous marrow rescue could reverse marrow aplasia and thyroidsupplement was essential to prevent thyroid damage. Although severeextramedullary toxicities were not seen, improvement in thepharmacokinetics of the radioconjugates will reduce significantly themarrow toxicity.

[0013] To date, a total of >95 patients have been treated with antibody3F8, and more than 120 imaging studies have been carried out ondifferent ongoing protocols. Among pediatric patients, no neuropathy hasbeen reported, either sensory or motor in nature. More than two thirdsof these patients mounted HAMA response, mostly low titer and notpersistent. There was no correlation of HAMA with toxicity.Nevertheless, in view of the neuropathy seen with other anti-G_(D2)antibodies 14.2a and 14.18 (similar in reactivity patterns to 3F8), wewant to improve the specificity to reduce side effects. All of theseclinical trials have been carried out using antibodies produced atMemorial Sloan-Kettering Cancer Center using guidelines of the Office ofBiologics Research and Review Center for Drugs and Biologics, Food andDrug Administration. For quality assurance, hybrdoma 3F8 was found to benegative for adventitious agents by MAP, S+L−, and XC plaque assays, aswell as negative for reverse transcriptase. MAP testing includedscreening for murine leukovirus, LCM virus isolation by intracerebralinoculation, murine saliva gland virus, mouse thymic virus, EDIM and LDHvirus isolations. Purified antibody (e.g. 3F8) had to pass MAP andsterility testing (bacteria, mycoplasma, and fungal cultures), rabbitpyrogen testing, as well as safety testing in mice and guinea pigs.Conjugation to 131I by the chloramine-T method was supervised by Dr.Ronald Finn and Dr. Steven Larson in the Department of Nuclear Medicine.Specific activity of iodine-131 was >600 mCi/ug iodide. Radiolabeledantibody 3F8 must have >50% binding by in vitro antigen bindingassay, >95% TCA precipitable and <3% free iodine by radio-thin layerchromatography. Periodic testing of radiolabeled antibodies wasperformed to ensure sterility as well as the absence of pyrogen.

[0014] Although the wide expression of G_(D2) in humanneuroectodermal-derived cancers (melanoma, small cell lung cancer,neuroblastoma, brain tumors, sarcoma, HTLV-1 leukemia, retinoblastomaand osteosarcoma) and the preliminary clinical studies of monoclonalantibodies to G_(D2) in radioimmuno-scintigraphy and radioimmunotherapyhave been encouraging, further optimization of antibodies for binding toG_(D2) would be desirable. It is an object of the present invention toprovide such optimized antibodies and the DNA sequences codingtherefore.

[0015] It is a further object of the invention to provide methods ofusing the optimized antibodies and DNA sequences in diagnostic assaysand therapeutic techniques.

SUMMARY OF THE INVENTION

[0016] The antibodies of the present invention are recombinant antibodyconstructs comprising the variable regions of the heavy and light chainsof anti-G_(D2) antibodies. These antibody constructs may be coupled to alabel such as a radiolabel or to a protein such as streptavidin orpro-drug converting enzymes for use in imaging or therapeuticapplications. The antibody constructs may also be transduced into Tcells to produce populations of T cells which target G_(D2)-producingtumor cells.

BRIEF DESCRIPTION OF THE DRAWING

[0017]FIG. 1 shows two alternative strategies for the use of theantibody constructs of the present invention in therapy.

DETAILED DESCRIPTION OF THE INVENTION

[0018] This invention relates to optimized antibodies to G_(D2), to DNAencoding such antibodies, and to the use of the antibodies and DNA indiagnostic assays and therapy. The antibodies of the invention areantibody constructs comprising the variable regions of the heavy andlight chains of anti-G_(D2) antibodies as a single chain Fv fragment.Single-chain Fv fragments (scFv) offer some of the best opportunities toachieve these results. ScFv technology utilizes molecular biologymethods to reduce antibodies to the minimal-required-unit of heavy andlight chain variable regions tethered by a peptide linker which can bedesigned with versatile side chains for radioconjugation.

[0019] The anti-G_(D2) scFvs shown in Tables 2 and 3 have been preparedusing the methods described in the examples. For 5F11 the orientationVH-VL is used for the scFv. For 3G6 the orientation VL-VH is necessary.Since the cDNA sequences of 3G6 and 3F8 are similar, we expect thebinding properties of both of these antibodies to be very similar if notidentical. Since our cloning strategies have been most successful with3G6, this has been the focus of our research instead of 3F8.Histidine-tag (His5) is inserted for ease of purification by Ni-columnand Myc-tag is inserted to facilitate detection. Myc-tag and E-tag canbe genetically removed if necessary for future clinical studies. ThesesvFv variants were constructed to test the effect of (1) thedetection-tag (E-tag or Myc-tag), (2) the presence of His5-tag, and (3)the position of His5-tag (carboxyl-end-terminal versus internal) on (a)antibody specificity, (b) affinity, and (c) ease of purification.

[0020] In order to increase the avidity of the scFv, we have synthesizedtwo scFv variants: (1) Cysteine residue at the carboxyl terminal of thescFv for dimerization (5FpoMCH of Table 2 and 3GpoMCH of Table 3): Freesulhydryl groups are blocked by acetylation and the monomer separatedfrom the dimer by size-exclusion chromatography FPLC on Sephadex HR75(PharTnacia). (2) Streptavidin at the carboxyl end for dimerization andtetramerization (5FpoStMCH of table 1 and 3GpoStMCH of table 2):Streptavidin is a homo-tetrameric protein that binds one biotin moleculeper subunit with a very high affinity (Kd=4×10−14). scFv-strep fusionproteins are expected to form tetramers with both antigen- andbiotin-binding activity They are expected to be stable over a wide rangeof pH and range of physiologic temperatures. TABLE 2 scFv Expression5FpcHE 5FphM 5FphHM 5FpoMCH 5FpoStMCH* Vector pCantab pHEN pHEN pOPEpOPE Tag E-tag + − − − − Myc-tag − + + + + His-tag + − + + + BacteriaHost phagemid XL1-blue XL1-blue XL1-blue − − phage hb2151 hb2151 hb2151JM109 JM109 Restriction sites: 5′ Ncol − + + − 5′ PvuII − − − + + 5′Sfil + − − − − 2′ NOT1 + + + + + Binding* G_(D2) elisa + + + + +ITLC + + + + + Western blot** + + + + + purification nd nd nd + +

[0021] TABLE 3 scFv Expression 3 GphM 3 GphHM 3 GPoMCH 3 GpoStMCH*Vector pHEN pHEN pOPE pOPE Tag E-tag − − − −/ Myc-tag + + + + His-tag− + + + Bacteria Host phagemid XL1-blue XL1-blue − − phage hb2151 hb2151JM109 JM109 Restriction sites: 5′ Ncol + + + + 5′ Sfil − − − − 3′NOTl + + + + Binding** G_(D2) elisa + + + + ITLC nd nd + nd anti-id ndnd + nd Western blot** + + + + Purification nd nd + nd

[0022] The 5F11-scFv, 3G6-scFv, 5F11-scFv-streptavidin,3G6-scFv-streptavidin DNA sequences are shown below, with the linkersequences between the scFv and the streptavidin shown in lower caseletters. SF11-scFv CAGGTGAAACTGCAGCAGTCAGGACCTGAACTGGTGNAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGANACAAATTCACTGAATACACCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTAACTACAAGCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAAGAGATACTACGGTCCCGTTTGCTTACTGGGTCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGGCAGCTCAAGTATAAGTTACATGCACTGGTACCAGCAGAAGCCTGTCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGTAGATGCTGCCACTTATTACTGCCATCAGCGGAGTAGTTACCCGCTCACGTTCGGTGCTGGGACACAGTTGGAAATAAAACGG 3G6-scFvAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTGGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCGAAACTGCTGATATACTCTGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCGCTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTGCAGGTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAATTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTTATGTCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGTCGGGGGGGTAACTACGGCTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 5F11-scFv-StreptavidinCAGGTGAAACTGCAGCAGTCAGGACCTGAACTGGTGNAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGANACAAATTCACTGAATACACCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTAACTACAAGCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAAGAGATACTACGGTCCCGTTTGCTTACTGGGTCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGGCAGCTCAAGTATAAGTTACATGCACTGGTACCAGCAGAAGCCTGTCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGTAGATGCTGCCACTTATTACTGCCATCAGCGGAGTAGTTACCCGCTCACGTTCGGTGCTGGGACACAGTTGGAAATAAAACGGgcggccgctggatccggtgctgctGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACAACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGC CATACATCACCATCATCAT3G6-scFv-streptavidinAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTGGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCGAAACTGCTGATATACTCTGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCGCTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTGCAGGTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAATTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTTATGTCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGTCGGGGGGGTAACTACGGCTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgcggccgctggatccggtgctgctGAAGCAGGTATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACAACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCGGATCCGAACAAAAGCTGATCTCAGAAGAAGATCTATGCATA CATCACCATATCAT

[0023] All these single chains have strings of 5 histidine residuesinserted at the carboxyl end for purification on the Nickel-column usingFPLC. Washing was done at 10 mM Imidazole and scFv peak elution at 500mM Imidazole. Further purification of scFv can be accomplished bysize-exclusion using Sephadex HR75 and superose 6 (hi-resolution). scFvcan be fuirther affinity purified by myc-affinity chromatography. 9E10is a hydridoma (ATCC) that secretes the antibody specific for themyc-tag. From ascites the 9E10 IgG1 antibody is purified by protein-Gaffinity chromatography and used for chemical coupling to sepharose 4B.

[0024] Recombinant antibody constructs according to the invention can becoupled to metal labels such as ^(99m)Tc for use in diagnostic imagingof G_(D2) expressing cells. For example, technetium can be chelated tothe antibody construct via a heterobifuinctional linker such assuccinimidyl-6-hydrazinonicotinate hydrochloride (SHNH). SHNH is used tosynthesize hydrazino-modified antibody. At a conjugation ratio of 1.8:1of SHNH to antibody, immunoreactivity was preserved. Tc(V) precursorscoupled readily and conveniently to the SHNH-modified protein to yieldthe desired 99mTc-radiolabeled conjugate. 99mTc-3F8 localized rapidlyand successfully to G_(D2)-positive xenografts. SHNH-modified scFvs canbe synthesized for conjugation to 99mTc using the techniques describedin Schwartz et al., Bioconjugate Chem 2:333-336 (1991), which isincorporated herein by reference.

[0025] Metal chelation to scFv can also be accomplished via thestreptavidin protein. The rationale of pretargeting usingscFv-streptavidin fusion proteins in radioimmunotherapy are 5-fold: (a)Large amounts of scFv can be used to saturate G_(D2) sites in vivo,without the accompanying blood and tissue toxicity from radioisotope,(b) radiolabel is injected at the time when the tumor-nontumor ratio ofscFv is maximal, (c) a radiolabeled ligand is chosen such that it bindswith high affinity (e.g. 111I-biotin binding to streptavidin) with fastblood-clearance, (d) a ligand construction where the isotope can bemodified to optimize microdosimetry (e.g. SHNH-biotin) (e) thescFv-streptavidin is a homo-tetramer, as such the antigen bindingavidity is greatly amplified especially for high-density antigens (e.g.G_(D2) on neuroblastoma). scFv-strep fusion proteins for both 5F11 and3G6 have been made and purified. Both in vitro and in vivo studies arebeing carried out to test the concept of pretargeting, where scFv-strepis first allowed to bind (or target) to G_(D2)-positive tumors throughthe scFv. After the excess or nonbinding scFv-strep is washed off (orcleared from the body), a radiolabeled-biotin ligand is allowed to bindto the streptavidin moiety. Different radiolabels can be coupled tobiotin using SHNH (^(99m)Tc) or DTPA (¹¹¹In or yttrium).

[0026] scFv coupled to technetium provides a safe, camera-ready isotope,with fast-decay and therefore easy disposal. ^(99m)-technetium isoptimal for imaging studies. Other isotopes can also be used, includinga positron-emitting technetium for PET imaging. Through the same sidechain SHNH, rhenium (a therapeutic beta-emitting radionuclide) may alsobe attached

[0027] The scFv and scFv-streptavidin of the invention are also usefulin a number of therapeutic applications, which is turn form aspects ofthe present invention In general, these approaches involveadministration of scFV coupled to a therapeutic or pre-therapeuticmoiety. For example, as shown in FIG. 1, ScFv-streptavidin (streptavidinbeing the pre-therapeutic moiety) is introduced into an organismsuspected of harboring G_(D2) expressing cells, where it binds to anysuch cells present. A therapeutic agent (X) bound to biotin is thenintroduced. Binding of the biotin the streptavidin results inlocalization of the chemotherapeutic agent X at the site of the G_(D2)producing cells. Other pre-therapeutic moieties include pro-drugconverting enzymes. Directly therapeutic moieties such as toxins canalso be used.

[0028] A second approach, also illustrated in FIG. 1, utilizes a vectorencoding ScFv is transduced into primary human lymphocytes (preferablyalong with a suicide gene such a HSV-TK). The transduced lymphocytes nowrecognize and target G_(D2), resulting in an immune response to theG_(D2)-producing cells.

[0029] The scFv or scFv-streptavidin can be incorporated in a fusionprotein with therapeutic agents such as toxins or pro-drug convertingenzymes, can be incorporated in a fusion protein with CD8 to facilitatethe formation of G_(D2)-targeted lymphocytes, or can be coupled to viralcoat proteins superantigen (SEA) to facilitate targeting of G_(D2)producing cells,

[0030] Direct conjugation of scfv or scFv-streptavidin to toxin replacesthe cell-binding domain of natural toxins with the scFv, which serves asa tumor binding domain specific to G_(D2) expressing cells.ScFv-ricin-A-chain and scFv-pseudomonas toxin have been successfullyconstructed for other scFv. This coupling is advantageously performed atthe DNA level, not at the protein level. For example, when the fusionprotein of the heavy chain, the light chain and the linker is created byoverlap PCR extension, a DNA coding for the toxin can also included, andthen expressed along with the scFv.

[0031] scFv and scFv-streptavidin can also be usefully combined in afusion protein with CD8. scFv-CD8 constructs can be transfected throughretroviral vector into human and mouse lymphocytes. Since these scFv arepermanently integrated into the cellular genome, these lymphocytesexpress scFv on their cell surface and through the CD8 cytoplasmicdomain become activated upon antigen binding. scFv facilitates thehoming of these cells to tumor sites, thus being effective in promotingboth the localization and killing of tumors. With a suicide gene,thymidine kinase, also transfected, these cells can now be turned on andoff as needed.

[0032] scFv-enzyme and scFv-enzyme-streptavidin conjugates can be usedto provide targeted drug therapy using a technique known as ADEPT(antibody directed enzyme prodrug-therapy). Suitable enzymes for thistechnique include carboxypeptidase G2, alkaline phosphatase, andβ-Lactamase. A prodrug derivative (e.g. cephalosporin derivative ofdoxo20) becomes activated to the active agent by the enzyme(beta-lactamase) targeted to the tumor by the scFv. Thus tumor cells areexposed to a high local concentration (up to 10-fold higher thanblood/tissue levels) of specific chemotherapeutic agents.

[0033] Integration of scFv (with or without streptavidin) into viralcoat proteins can be used to retarget these viruses in vivo. Theseviruses include adenovirus, retrovirus and herpes virus.

[0034] Superantigen (SEA) can stimulate T cells without the requirementof MHC.21 ScFv-SEA and scFv-streptavidin-SEA can target T cells to lyseantigen-positive MHC-class II-negative human tumor cells. SEA has beencloned (Betley et al: J. Bacteriology 170: 34-41, 1988) and the cDNA isavailable for making fusion proteins.

EXAMPLE 1

[0035] 5F11 hybridoma cells were processed for mRNA using a commerciallyavailable kit (Quick Prep Micro MRNA Purification, pharmacia Biotech)following the procedures outlined by the manufacturer. Briefly,hybridoma cells were cultured in ROPMI-1640 medium supplemented with 10%calf serum, 2 mmol/L L-glutamine (Sigma), 100 U/L penicillin and 100ug/ml streptomycin sulfate (Sigma). The cell cultures were maintained at37° C. under a water-saturated atmosphere of 5% CO₂. 5×10⁶ cells werepelleted by centrifugation at 800 g and washed once with RNase-freephosphate buffered saline (pH 7.4), The recentrifuged cells were yseddirectly in the extraction buffer. Poly(A)+RNA was purified by a singlefractionation of oligo(dT)-cellulose and then eluted with elutionbuffer. The mRNA sample was precipitated for 1 hour at 100 ug glycogen,40 ul of 2M potassium acetate and 1 ml absolute ethanol at −20° C. Thenucleic acids were recovered by centrifugation at 10,000 g for 30minutes, The sample was evaporated until dry and dissolved in 20 ulRNase-free water.

[0036] The mRNA preparation was used in the construction of the 5F11scFv gene using the Mouse ScFv Module/Recombinant Phage Antibody System(Pharmacia Biotech). 5 ul of the mRNA preparation was reversetranscribed in a total volume of 11 ul of reaction mixture and 1 ul DTTsolution for 1 hour at 37° C. For PCR amplification of immunoglobulinvariable region, light primer mix and the heavy primer set were addedrespectively to generate quantities of the light (325 bp(and heavy (340bp) chains. Following an initial 5 minute dwell at 95° C. , 5 U AmpliTaq DNA polymerase (Perkin Elmer) was added. The PCR cycle consisted ofa 1 min denaturation step at 94° C. , a 2 min. annealing step at 55° C.and a 2 min extension step at 72° C. After 30 cycles of amplification,PCR derived fragments were purified using glassmilk beads (Bio 101 Co.)and evaluated by electrophoresis on 1/5% agarose gel in TAE buffer withethidium bromide visiualization. For the assembly and fill-in reaction,both purified heavy chain and light chain fragments were added to abappropriate PCR mixture containing linker-primer, dNTPs, PCR buffer andAmpli Taq DNA polymerase. Denaturation was performed at 94° C. for 1minute, followed by a 4 minute annealing reaction at 63° C. The heavyand light DNA were joined into a single chain with linker DNA after 7thermocycles. Using this single chain DNA as a template and restrictionsite primers (RS primers) containing either SfiI or NotI restrictionsites, secondary PCR amplification was carried out for 30 cycles toamplify the ScFv DNA and add the restriction sites. This introduced theSfiI restrictions site at the 5′-end of the heavy chain and the NotIrestriction site at the 3′-end of the light chain. Amplified ScFv DNAwas then purified by glassmilk beads and digested with SfiI and NotI.

[0037] Purified scFv DNA was inserted into the pCantab 5e vector(Pharmacia Biotech) by ligation as SfiI/NotI sites in the vector.Competent E. coli XL 1-Blue cells were transformed with pCantab 5Ephagemid containing the ScFv DNA following the method outlined inStratagene protocols. For rescue of a recombinant phage antibodylibrary, the helper phage M13 K07 was added.

[0038] Antibody-producing recombinant phage were selected by panningusing the method of Ditzel, PNAS USA 91: 3710-3714 (1994) with slightmodifications. 20 ul of GD2 (1 ul/ml) dissolved in ethanol were directlycoated on a 96-well polystyrene plate and dried at rom temperature. Then100 ul of the supernatant containing the phage library was added to eachwell and incubated for 2 hours. The plate was then washed 10 times withPBS containing 0 05% BSA to remove nonspecifically bound phage.Antibody-positive recombinant phage captured by the G_(D2) antigen waseluted with 0.1 M HCl (pH 2.2 with solid glycine and 0.1% BSA) andneutralized with 2M Tris solution. Selected phage was then re-panned fortwo additional cycles to further enrich the GD2-binding recombinantphages.

[0039] The selected phage was used to reinfect E coli XL1-Blue cells.Clones were grown in 2XYT medium containing ampicillin (100 ug/ml) and1% glucose at 30° C. until an OD₆₀₀ of 0.5 was obtained. Expression ofScFv antibody was induced by changing to a medium containing 100 uM IPTGand incubating overnight at 30° C. The supernatant obtained from themedium by centrifugation was directly added to a plate coated with GD2.The pellet was resuspended in PBD containing 1 mM EDTA and incubated onice for 10 minutes. The periplasmic soluble antibody was collected bycentrifugation again and added to the plate. After incubating at 37° C.for 32 hours, anti-E Tag antibody (Pharmacia Biotech) was used tospecifically screen the binding of the ScFv fragment.

[0040] For construction of the 5FpoStMCH vector which contains the5F11-scFV-streptavidin plasmid DNA, plasmid DNA from the 5F11-scFv inpCantab 5E vector (Pharmacia Biotech) was purified and amplified by PCRusing two specially designed primers S6 and 318s. S6 contains a NotIrestriction site and 318s contains a PvuII restriction site so thatamplified DNA can be restriction digested and inserted in the pSTEvector (Dr. Dubel, German Cancer Center). The resulting vector 5FpoStMCHis the 5F11-scFv-streptavidin construct. The streptavidin was digestedwith BamHI, leaving the scFV 5FpoMCH.

EXAMPLE 2

[0041] Supernatant, periplasmic extract and cell extract from positiveclones were fractionated on unreduced SDS-PAGE 12% SDS-polyacrylamideslab gels and buffer prepared according to Laemmli (1970).Electrophoresis was performed at 100V for 45 min. After completion ofthe run, western blot analysis was carried out as described by Towbin(1979). The nitrocellulose membranes were blocked by 1% nonfat milk inTBS solution for 1 hour and incubated with anti-E Tag antibody for 1hour at room temperature. After incubating with HRP-conjugated goatanti-mouse Ig (Fisher Co.), the membrane was detected by ECL System(Amersham). The results showed a protein band with an apparent molecularmass of 31KD using anti-E Tag antibody which recognizes the sequenceGAPVPVPDPLEPR. The same protein was not detected in control cells nor incells without IPTG treatment to induce expression of the scFV.

EXAMPLE 3

[0042] Immunostaining thin layer chromatography was performed underconditions similar to those described by Tai et al (1987) GD2, GD3,GD1a, GD1b, GM2, GT1b and GL1000 were dissolved in ethanol and spottedon an HPTLC plate. The supernatant from ScFv 5F11 clone and 5F11hybridoma cells were incubated with the spread plate. Immunostaining wasvisualized with the use of o-phenylenediamine dihydrochloride (Sigma).GD2 antigen was detected by both the ScFv supernatant and the 5F11 Mab,which appeared similar in specificity. Cross-reactions of the ScFvantibody with other glycolipids was not detected.

EXAMPLE 4

[0043] In constructing 3G6-scFv, the orientation VH-VL did not produce afunctional scFV. Therefore the orientation VL-VH was used. cDNA of VHand VL of 3G6 hybridoma were linked through a custom built linker andinserted into the pHEN vector (DR. Greg Winter). NcoI and NotIrestriction sites were built into the VH and VL linkers so that the scFVcan be digested with these enzymes for insertion in the pSTE vector.Clone 7 was chosen and called 3GpoStMCH. Digestion of the streptavidinposition of the gene left behind 3G6-scFv, now called 3GpoMCH.

[0044] The following references are cited above, and are incorporatedherein by reference.

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[0047] 3. Traylor T D, Hogan E L: Gangliosides of human cerebralastrocytomas. J Neurochem 34:126-131, 1980

[0048] 4. Ye J N, Cheung N K V: A novel O-acetylated gangliosidedetected by anti-G_(D2) monoclonal antibodies. Int J Cancer 50-197-201,1992

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[0050] 6. Longee D C, Wikstrand C J, Mansson J E, et al:Disialoganglioside G_(D2) in human neuroectodermal tumor cell lines andgliomas. Acta-Neuropathology (Berl) 82:45-54, 1991

[0051] 7. Hoon D S, Banez M, Okun E, et al: Modulation of human melanomacells by interleukin-4 and in combination with gamma-interferon oralpha-tumor necrosis factor. Cancer Res 51:2002-2008, 1991

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[0053] 9. Saito M, Yu R K, Cheung N K V: Ganglioside G_(D2) specificityof monoclonal antibodies to human neuroblastoma cell. Biochem BiophysRes Comm 127:1-4, 1985

[0054] 10. Lammie G A, Cheung N K V, Gerald W, et al: Ganglioside G_(D2)expression in the human nervous system and in neuroblastomas—animmunohistochemical study. Int J Oncol 3:909-915, 1993

[0055] 11. Sariola H, Terava H, Rapola J, Saarinen U M: Cell-SurfaceGanglioside G_(D2) in the Immunohistochemical Detection and DifferentialDiagnosis of Neuroblastoma. AJCP 96:248-252, 1991

[0056] 12. Saarinen U M, Sariola H, Hovi L: Recurrent DisseminatedRetinoblastoma Treated by High-dose Chemotherapy, Total BodyIrradiation, and Autologous Bone Marrow Rescue. Am J PediatrHematol/Oncol 13:315-319, 1991

[0057] 13. Heiner J, Miraldi F D, Kallick S, et al: In vivo targeting ofG_(D2) specific monoclonal antibody in human osteogenic sarcomaxenografts. Cancer Res 47:5377-5381, 1987

[0058] 14. Chang H R, Cordon-Cardo C, Houghton A N, et al: Expression ofdisialogangliosides G_(D2) and GD3 by human soft tissue sarcomas. Cancer70:633-638, 1992

[0059] 15. Cheung N K, Neely J E, Landmeier B, et al: Targeting ofganglioside G_(D2) monoclonal antibody to neuroblastoma. J Nuci Med28:1577-1583, 1987

[0060] 16. Yeh S D, Larson S M, Burch L, et at: Radioimmunodetection ofneuroblastoma with iodine-131-3F8: Correlation with biopsy,iodine-131-Metaiodobenzylguanidine (MIBG) and standard diagnosticmodalities. J Nucl Med 32:769-776, 1991

[0061] 17. Miraldi F D, Nelson A D, Kraly C, et al: Diagnostic imagingof human neuroblastoma with radiolabeled antibody. Radiology161:413-418, 1986

[0062] 18 Grant S C, Kostakoglu L, Kris M G, et al: Imaging of smallcell lung carcinoma with the monoclonal antibody 3F8. Proc Am Soc ClinOncol 10:265, 1991 (abstract)

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1. A recombinant single chain polynucleotide comprising a regionencoding the variable region of the light chain of an anti-G_(D2)antibody linked to a region encoding the variable region of the heavychain of an anti-G_(D2) antibody.
 2. The recombinant polynucleotide ofclaim 1, further comprising a region encoding an additional protein. 3.The recombinant polynucleotide of claim 1, wherein the additionalprotein is streptavidin.
 4. The recombinant polynucleotide of claim 1,wherein the additional protein is a drug-converting enzyme.
 5. Arecombinant single chain peptide comprising the variable region of thelight chain of an anti-G_(D2) antibody linked to the variable region ofthe heavy chain of an anti-G_(D2) antibody.
 6. The peptide according toclaim 5, wherein the peptide is labeled with a radiolabel.
 7. Thepeptide according to claim 6, wherein the radiolabel is ^(99m)Tc.
 8. Thepeptide according to claim 5, wherein the peptide further comprises adrug-converting enzyme.
 9. The peptide according to claim 5, wherein thepeptide further comprises streptavidin.
 10. The peptide according toclaim 5, wherein the peptide further comprises CD8.
 11. T cellsexpressing a recombinant single chain peptide comprising the variableregion of the light chain of an anti-G_(D2) antibody linked to thevariable region of the heavy chain of an anti-G_(D2) antibody.
 12. Amethod for assaying for the presence of cells expressing G_(D2) intissue comprising combining the tissue with a recombinant single chainpeptide comprising the variable region of the light chain of ananti-G_(D2) antibody linked to the variable region of the heavy chain ofan anti-G_(D2) antibody and a detectable label.
 13. A method fortargeted delivery of a therapeutic agent to cells expressing G_(D2) intissue comprising combining the tissue with a recombinant single chainpeptide comprising the variable region of the light chain of ananti-G_(D2) antibody linked to the variable region of the heavy chain ofan anti-G_(D2) antibody and a therapeutic or pre-therapeutic moiety. 14.The method according to claim 13, wherein the pre-therapeutic moiety isa pro-drug converting enzyme.
 15. The method according to claim 13,wherein the pre-therapeutic moiety is streptavidin.
 16. The methodaccording to claim 13 wherein the therapeutic moiety is a toxin.